Miniaturization of chemical analysis is a highly active area of intense scientific research. Much of the research is driven by the health and life sciences, where miniaturization has the capacity to revolutionize the diagnosis and treatment of disease [Yager et. Al Nature 2006, 442, 412-418; Chin, Linder, Sia Lab Chip, 2007, 7, 41-57]. Central to this theme is the miniaturization of processes and procedures that occur in conventional chemical and biological laboratories. These activities include sampling, storage, sample treatment, separation, detection, and analysis. Miniaturization uses less sample, offers superior detection sensitivity, and has the potential to greatly reduce the costs of laboratory environment, labor, and materials. Efforts at miniaturization have focused primarily on the implementation of so-called microfluidic “lab-on-chip” devices [Chin, Linder, Sia, Lab Chip, 2007, 7, 41-57], although more conventional methods, such as lateral flow chromatography, have also been reduced in scale [Yager et. Al Nature 2006, 442, 412-418].
A particularly promising analytical technology for medical diagnostics from biological tissues and fluids is liquid chromatography coupled to mass spectrometry (LC-MS) [Hoofnagle, Clin. Chem. 2010, 56, 161-164; Anderson Clin. Chem. 2010, 56, 177-185]. LC-MS is a powerful method, but requires a highly complex analytical system. Current state-of-the-art practice requires expert level training of staff, together with a significant investment in laboratory infrastructure. Centralized laboratory resources coupled together with remote sampling of patient populations is a common solution to meet these multiple requirements for clinical analysis.
Electrospray ionization is a well-established method to ionize liquid samples for chemical analysis by mass spectrometry. Nanoelectrospray ionization, also referred to as nanospray, is a miniaturized low-flow and low-volume variant of electrospray ionization. Nanospray has been shown to offer superior sensitivity and selectivity compared to conventional electrospray ionization. Nanospray is the path to chemical miniaturization for mass spectrometry.
Various methods have been developed for using nanospray for either off-line analysis of individual discrete liquid samples, or on-line analysis of flowing liquid streams, e.g. the effluent from liquid chromatography. Off-line nanospray, which is the subject of this invention, is also referred to in the prior art as static nanospray.
Diagnostic testing places strict demands on the mass spectrometric analytical system. It is highly desirable for a diagnostic analysis to have no interference from one sample analysis to the next (also known as zero “carry over”). In a miniaturized analytical system, where the surface-area-to-volume ratio is high, a non-redundant fluid path is preferred. Thus a nanospray system having a non-redundant fluid path is preferable for application in a clinical setting.
A commonly employed apparatus for off-line nanospray utilizes a nanospray emitter fabricated from a tube, typically 1-2 mm inside diameter (ID), having a finely tapered end in which the ID tapers to a 1-5 μm ID orifice. The tapered end is referred to as the proximal end. A liquid aerosol emits from the proximal end during the electrospray process. Such emitters are generally fabricated from borosilicate glass, fused-silica, or fused quartz, although other materials including polymers and metals have been employed. The non-tapered end is referred to as the distal end, and is the end of the emitter to which sample is typically loaded. The nanospray emitter is commonly coated with an electrically conductive metal or polymer film. The coating covers the entire outer surface of the emitter and makes contact with the liquid sample, typically at the proximal end, although contact at the distal end is also feasible. The purpose of the electrically conductive coating is to establish a potential difference (typically 1000-5000 V) between the liquid inside the emitter and the inlet of the mass spectrometer.
A significant challenge for successful off-line nanospray is three-fold: (A) Samples must be fairly clean and concentrated prior to use. (B) Sample volumes should be low, preferably less than 10 uL, and more preferably less than 5 uL. (C) Sample transfer of microliter sample volumes into the emitter is time consuming, risky and difficult. Low volume samples for analysis are loaded into the emitter in one of four ways: Injection from a syringe using a fine needle, injection from a hand pipette using a finely tapered plastic tip, transfer from another (glass) capillary tube into the nanospray emitter by means of a centrifuge, or capillary action from a sample reservoir.
These methods are typically time consuming, expensive, and/or require a great deal of hand manipulation and fine motor skills. The glass nanospray emitters are fairly delicate and fragile. The small ID's for the emitters (<2 mm) require the use of small diameter liquid injection tools. Expert level training is usually required for successful application of the technique. With perhaps the exception of method (3), these methods are poorly suited to low-cost, automated or high-throughput laboratory procedures.
Thus there is a significant need for a miniaturized system having a non-redundant fluid path for the isolation, storage, purification, and analysis of samples by nanospray ionization mass spectrometry. It is particularly desirable that the system be easy-to-use, low cost, and offer high throughput. It should enable discrete sampling and storage of samples in the liquid or dry state, remote from the analytical laboratory. It's use should require a minimum of specialized laboratory equipment, preferably limited to the apparatus commonly found in a clinical or hospital environment.
The present invention address these issues by applying desirable aspects of the centrifuge transfer method to the integration of nanospray with efficient sample preparation methods. In a preferred embodiment, the invention combines and couples nanospray to trap-and-elute sample preparation by solid phase extraction (SPE).
SPE is a generic term for a wide variety of well-established and well-known methods for the isolation and purification of target chemical compounds present in simple or complex mixtures from fluid (liquid or gaseous) samples. For example, U.S. Pat. Nos. 3,953,172; 4,142,858; 4,270,921; 4341635; 4650784; 4774058; 4820276; 5266193; 5279742; 5368729; 5391298; 5595653; 5415779; 5538634 describe methods and devices for carrying out solid-phase extraction from liquid or gaseous samples.
SPE is based on the extraction and concentration of target compounds present in the liquid (or gas) sample onto the surface of a high-surface area solid substrate, referred to as the solid phase. It is dependant on the affinity of a target compound for the specific surface chemistry of the solid phase. The solid phase is typically wetted by the sample, but is not soluble in the sample. High surface area solid phases are available in a wide variety of surface chemistry including hydrophilic, hydrophobic, and cationic (positively charged) and anionic (negatively charged).
The solid phase is typically porous in nature so that liquid samples may pass though the solid phase when a pressure difference is applied across the substrate. The pressure differential can be provided by: liquid pumps, pressurized syringes, the application of gas pressure or vacuum, or by centrifugation of liquid through the solid phase. When the liquid passes through the solid phase target compounds having a high affinity for the surface will be trapped and retained on the substrate surface. The volume of liquid that can be passed through the solid phase is generally unrestricted, and is typically many times (>10×) the volume of the substrate. This ratio provides the capability of concentrating the target compound from a large volume of liquid onto a small volume of substrate. A small volume solid phase also ensures that a smaller volume of liquid may be used for extraction.
The target compound is typically subsequenty removed from the solid phase by the process of elution. A volume of a liquid, referred to as the eluent, is chosen so that the target compound is highly soluble in the eluent. When the solid substrate is brought into contact with the eluent, the target compound is extracted from the solid phase surface, and dissolved in the eluent. By using a volume of eluent that is smaller than the original sample, the target compound will then be present in the eluent at a higher concentration than that of the original sample liquid. The volume of eluent is preferably much less (<one-tenth) than the volume of the original sample.
Assuming that 100% of the target compound in the original liquid sample is trapped by the solid phase, and that 100% of the trapped compound is extracted by the eluent; the practical concentration of target compound provided by SPE is dependant on the volume ratio of sample-to-eluent. In practice the degree of trapping and elution is less than 100%. The volumetric ratio of sample-to-eluent represents a practical upper limit for compound concentration with conventional SPE methodology.